Clusters of non-heme iron and inorganic sulfur are ubiquitous electron carriers in fundamental life process such as respiration, photosynthesis, fermentation, and nitrogen fixation. However, the diversity of the biological roles for iron-sulfur clusters has only recently become apparent. In addition to being a major determinant of protein structure and stability, functional roles for iron-sulfur clusters now include substrate binding and activation in numerous redox and non-redox enzymes, generation and/or stabilization of radical intermediates, control of gene expression in iron regulation, and electron and/or iron storage. The majority of these roles require site-specific reactions at iron-sulfur clusters. The overall objective of this research program is to understand the diverse functions and properties of iron-sulfur clusters at the molecular level by studying a simple iron-sulfur protein, the ferredoxin from the hyperthermophilic archaeon, Pyococcus furious, and organism that grows optimally at 100 C. P. furious ferredoxin (M=7,500) contains a single [Fe4S4] cluster. It is remarkable both for this extreme thermal stability (at least 12 hours at 95 C) in being the only example of 4Fe-ferredoxin that has non-cysteinyl ligation at a specific iron site, and in the ease of quantitative removal of this iron to give a [Fe3S4] cluster. This not only facilitates the formation of heterometallic clusters, [MFe3S4], where M is a first row transition metal it also allows investigations into site-specific chemistry at both homometallic and heterometallic iron-sulfur clusters in a biological environment. Moreover, it is the only 4Fe-ferredoxin whose gene has been cloned and successfully expressed at the holoprotein in Escherichia coli. A comprehensive research program is proposed involving the use of biophysical techniques to investigate native and site-directed mutant forms of P. furious ferredoxin. The combination of nuclear magnetic resonance, X-ray crystallography, and calorimetry will be used to determine protein structure and stability. The structural, redox, and electronic properties of the iron-sulfur center will be assessed using a range of complimentary techniques; nuclear magnetic resonance, X-ray crystallography, electron paramagnetic resonance, UV/visible/near-IR absorption and natural and magnetically-induced circular dichroism, resonance Raman, Mossbauer, electron nuclear double resonance, and x-ray absorption. The results will provide unique insights into the factors that determine protein "hyperthermostability" and the structural and functional diversity of biological iron-sulfur clusters.